Due to its extensive penetrance of the human population, Mycobacterium tuberculosis (Mtb) remains a serious health risk to those individuals living with HIV. TB vaccine development programs are hampered by our poor understanding of the immune mechanisms underpinning disease progression. What we propose in this application is the utilization of Mtb reporter strains to provide a functional readout of microbial fitness and replication to enable us to identify and characterize those phagocytes that restrict bacterial growth (controllers) versus those phagocytes the promote bacterial growth (permissive) to understand the basis of disease progression in human tuberculosis. Our hypothesis is that Mtb reporter strains represent a novel route to the identification of the phagocyte populations that best restrict or promote bacterial replication, and that defining these cell populations will provide a rational framework for understanding immune control of tuberculosis. Aim 1: Development and validation of Mtb reporter strains and challenge models in the murine system. We will (a) exploit Mtb reporter strains to identify permissive and controller phagocyte populations; (b) optimize an ex vivo infection protocol for cells recruited to Mtb-infected mouse lung, to be used on NHP granulomas, and human airway and granuloma phagocytes; and (c) perform RNASeq profiling on the different phagocyte populations defined by the Mtb reporter strains for phenotypic analysis and to generate a panel of candidate cell surface markers for NHP and human ex vivo challenge studies. Aim 2. Functional characterization of phagocyte subsets in NHP pulmonary granulomas. We will perform parallel analysis of NHP phagocyte populations infected with reporter bacterial strains following the isolation of individual granulomas from infected monkey lungs. We propose comparable phenotypic characterization using biased and unbiased methods to functionally identify permissive and controller phagocyte populations in NHP tuberculosis in in vivo infections and ex vivo challenge experiments. Specific Aim 3: Determination of human phagocyte phenotypes through ex vivo challenge with Mtb reporter strains. In addition, in collaboration with Drs. Henry Mwandumba (Malawi) and Alasdair Leslie (South Africa) we will determine the functional phenotypes of human airway and human TB granuloma phagocyte subsets by probing the cells ex vivo with the fluorescent Mtb reporter strains.